Peptides and polypeptides useful for regenerating the nervous system

ABSTRACT

The invention concerns in particular novel peptides and polypeptides comprising at least the following sequence in amino acids: -W-S-A1-C-S-A2-C-G- in which A1 and A2 are amino acid sequences comprising 1 to 5 amino acids, useful as medicines in therapeutic treatments involving the regeneration of the nervous system cells, for treating neuroblastomas, and also useful as additives in the culture of nervous cells.

This application claims priority to French Application 97-0916 filed onJul. 16, 1997 and international application PCT/FR98/01556, filed onJul. 16, 1998, designating the United States of America, and publishedon Jan. 28, 1999, in the French language in accordance with PCT Article21(2) as WO 99/03890.

The present invention relates in particular to novel peptides andpolypeptides useful in particular as medicines in therapeutic treatmentsinvolving the regeneration of the nervous system cells, for treatingneuroblastomas, and also useful as additives in the cultures of nervecells.

Many proteins comprising repeating units which are called thrombospondintype I units (TSRs) have been identified during the past few years. Itcan be said that these proteins have highly varied activities dependingon the biological system in which they are involved. There may bementioned, as the best studied and therefore the best known examples,the CS proteins (of circumsporozoite) which allow binding to the hepaticcells of the agent for the propagation of malaria, the plasmodiumfalciparum sporozoite (WO 94/06646) and the thrombospondin secreted bythe blood platelets which are involved in the phenomena of thrombosisand angiogenesis (EP 443 404).

In fact, this thrombospondin type 1 unit (TSR) comprises, in all theproteins studied so far and previously mentioned, about 60 amino acids(AA) some of which, like cysteines (C), tryptophans (W), serines (S),glycines (G), arginines (R) and prolines (P) are highly conserved (seebelow the alignment of these conserved AAs in a few proteins).

Some synthetic peptides, deduced from these TSR units, have valuablebiological properties. Thus, the CSVTCG (SEQ ID NO:21) units allow theadhesion of the plasmodium sporozoites to the hepatic cells, the CSVTCG(SEQ ID NO:21) and WXXW (SEQ ID NO:22) units allow cellular attachmentin other biological models, BBXB (SEQ ID NO:23) (B being a basic aminoacid) binds heparin and finally WSXWS (SEQ ID NO:24) binds certaingrowth factors.

F-spondin has been described and its sequence has been aligned with thatof thrombospondin in Klar et al., (1992), Cell, 69, 95–110.

The general characteristics of SCO-spondin are described in particularin the article by Monnerie et al. (submitted) and the article by Gobronet al., (1996), Journal of Cell Science, 109, 1053–1061, 1996. Inparticular, the alignment of the sequence of SCO-spondin has revealedhomologies with proteins such as thrombospondin 1 and 2 (see sequence,alignment page 1057 of Gobron et al., (1996), J. of Cell Science 109,1053–1061, incorporated into the description by reference).

The novelty of the present invention consists in the identification andthe selection of a novel peptide which is active in the regeneration ofthe nervous system, whose sequence is derived from one of the TSRs ofSCO-spondin.

More particularly, the present invention relates to a peptide orpolypeptide having the formula:-W-S-A₁-C-S-A₂-C-G- (SEQ ID NOS: 1 and 25–49)

-   -   in which A₁ and A₂ are amino acid sequences comprising 1 to 5        amino acids, with the exception of the peptides or polypeptides        having one of the following sequences        -W-S-P-C-S-V-T-C-G- (SEQ ID NO: 2)        -W-S-S-C-S-V-T-C-G- (SEQ ID NO: 3)        -W-S-Q-C-S-V-T-C-G- (SEQ ID NO: 4)

It should be recalled that in the description as a whole, “amino acid”is understood to mean both the natural amino acids and the non-naturalamino acids. “Natural amino acid” is understood to mean the amino acidsin the L form which can be found in natural proteins, that is to sayalanine, arginine, asparagine, aspartic acid, cysteine, glutamine,glutamic acid, glycine, histidine, isoleucine, leucine, lysine,methionine, phenylalanine, proline, serine, threonine, tryptophan,tyrosine and valine. However, the present invention also relates to thenon-natural amino acids, that is to say the preceding amino acids intheir D form, as well as the homo forms of some amino acids such asarginine, lysine, phenylalanine and serine or the nor forms of leucineor valine.

It is also possible to envisage using other amino acids such as, forexample:

Abu: alpha-aminobutyric acid Agm: agmatine Aib: alpha-aminoisobutyricacid F-trp: N-formyl-trp

-   sarcosine-   statine-   ornithine-   desaminotyrosine.

Desaminotyrosine is incorporated at the N-terminal end whereas agmatineand statin are incorporated at the C-terminal end of these peptides.

Preferably, the peptides according to the present invention A₁ isproline or X₁-W-X₂-X₃ (SEQ ID NOS: 5 and 50–59) where X₁, X₂, X₃ arechosen, independently of each other, from G, S and C, that is to saysmall amino acids.

Still preferably, A₁ is X₁-W-S-X₃ (SEQ ID NOS: 6 and 60–64) and A₂ ischosen from RS, VS and VT.

The reasons for these choices will emerge on reading some examples.

Preferably, the polypeptide according to the present invention has thefollowing structure:-W-S—X₁-W-S-X₂-C-S-A₂-C-G- (SEQ ID NOS: 7 and 89–96)

The preferred peptide has the following structure:-W-S-G-W-S-S-C-S-R-S-C-G- (SEQ ID NO: 8)

Preferably, the peptides and polypeptides according to the presentinvention will have the following structure:Y-W-S-A₁-C-S-A₂-C-G-Z (SEQ ID NOS: 9 and 97–168)

-   -   in which Y and Z constitute the N- and C-terminal ends of the        peptide, or comprise amino acid chains having less than 6 amino        acids, or comprise chains of compounds which are not amino        acids.

This corresponds to the peptide per se or to a peptide in which the Zand Y ends enhance the pharmacological activity or ensure a betterpenetration or bioavailability of the active ingredient; thus, it ispossible to envisage in the Y and Z ends the use of hydrophiliccomponents which make it possible, where appropriate, to cross certainbiological barriers, or alternatively, on the contrary, to envisage morehydrophilic sequences which will allow a better solubilization of theproducts involved.

Finally, the modification of the ends can facilitate the incorporationof these products into particular galenic forms such as, for example,liposomes or microparticles.

The present invention also relates to DNA expression vectorscharacterized in that they are capable of expressing the precedingpeptides or polypeptides.

The DNA sequences encoding the preceding peptides or polypeptides can beeasily determined from amino acid sequences or based, for example, onthe natural sequences as will be described in the present application.

The vectors for administration may consist of naked DNA vectors, plasmidvectors, viral vectors or alternatively synthetic vectors.

These are known technologies which will not be described in detail.

The use of these expression vectors makes it possible to express in situthe peptides or polypeptides involved and, in some cases, is likely toenhance their activity.

Constructs will of course be chosen which exhibit, if possible,specificity for the nerve cells, since they are the preferred targetsfor the polypeptides according to the present invention.

The peptides and polypeptides according to the present invention may beprepared by any appropriate method, in particular they may be obtainedby chemical synthesis, but it is also possible to obtain them by thebiological route using in particular the vectors mentioned above inappropriate cell cultures.

It should in fact be noted, in this regard, that the polypeptides andpeptides according to the present invention may be provided indeglycosylated or glycosylated form if necessary. It should also benoted that in some cases and depending on the method of preparation, itmay be necessary to renature some tertiary structures of the peptide.

Finally, the polypeptides according to the present invention can be moreparticularly used for the manufacture of a medicine with the aim ofbeing administered in vivo, in particular in all pathological conditionsand traumas requiring regeneration of the nervous system cells, and moreparticularly of their outgrowths and synapses.

These may be pathological conditions or traumas in whichneurodegeneration is observed, but they may also be pathologicalconditions or traumas in which the regeneration of the central nervoussystem, in particular of the axons, or of the peripheral nerves isnecessary.

Among the neurodegenerative pathological conditions in which thecompounds according to the present invention may provide a support,there may be mentioned in particular Alzheimer's disease, multiplesclerosis, Parkinson's disease and the different types of myopathies.

As regards the regeneration of the neuronal outgrowths, in particular ofthe axons, this may involve in particular accident- or trauma-typeproblems (section of the spinal cord or of the peripheral nerves).

Likewise, the compounds according to the present invention may be usedas additives in certain cell cultures with the same effects as thosementioned above on the growth of cells.

More particularly, the compounds according to the present inventionincrease neuritic growth (including the axons) in the cerebral cortexneurons. Inhibition of aggregation and defasciculation of the neuritesare noted on the spinal cord neurons and an increase in synapticcontacts is also noted.

“Neuritic growth” is defined as an extension, that is to say growth ofthe neuron outgrowths, whether the dendritic or axonal outgrowth.

“Aggregation” is defined as a grouping together of the cells forming acluster.

“Defasciculation” is defined as the result of a decrease in adhesivitybetween neurites, leading to a loose network of neuronal outgrowths.

“Synaptic contact” is defined as the capacity for a neuronal cell tocommunicate with another cell, it being possible for the latter to alsobe neuronal.

In another aspect of the present invention, said peptides orpolypeptides may be useful for inducing regression of tumorigenicityduring a neuroblastoma.

The nomenclature used to describe the sequence of the present peptide isthe international nomenclature using the three-letter code or theone-letter code and where the amino-terminal end is presented on theleft and the carboxy-terminal end is presented on the right.

The compositions according to the present invention may be provided inany customary form for pharmaceutical administration, that is to say forexample forms for liquid administration in a gel or any other supportallowing, for example, controlled release.

Among the compositions which may be used, there should be mentioned inparticular the injectable compositions more particularly intended forinjections into the meningeal and subarachnoidal spaces.

The most active peptide according to the present invention has thefollowing formula:Trp-Ser-Gly-Trp-Ser-Ser-Cys-Ser-Arg-Ser-Cys-Gly (SEQ ID NO: 8)

It is soluble in basic aqueous medium, has a molecular weight of 1301 Daand has an amino acid composition of:

N N (%) MW MW (%) C Cys Cysteine 2 16.7 206 15.8 G Gly Glycine 2 16.7114 8.8 R Arg Arginine 1 8.3 156 12.0 S Ser Serine 5 41.7 435 33.4 W TrpTryptophan 2 16.7 372 28.6

It was obtained by solid phase chemical synthesis.

However, as was indicated above, it can be obtained by geneticengineering using a host-vector system comprising DNA encoding thepeptide taking into account, for example, the degeneracy so as toproduce it in a large quantity.

The cDNA sequence encoding the peptide may be 20 presented in thefollowing manner (SEQ ID NO: 10):

5′ TGG WSN GGN TGG WSN WSN TGY WSN MGN WSN TGY GGN 3′ A= Adenosine     W = A or T C = Cytosine      S = G or C G= Guanosine     Y = C or T T = Thymidine     M = A or C N = A, C, G or T

The peptide thus obtained was identified by microsequencing, HPLCanalysis, mass spectrometry and sequencing of the complementary DNA.

It is on this peptide (SEQ ID NO: 8) that the experiments describedbelow were carried out.

EXAMPLE 1 Effect of the Peptide SEQ ID NO: 8 on the Growth of theNeurons

Materials and Method

Dissociated Cell Cultures of Cerebral Hemispheres of 8-Day Old ChickenEmbryos

The neuronal cultures are obtained from 8-day old chicken embryos. Thecerebral hemispheres, after removing the meninges, are cut into smallpieces and enzymatically dissociated with 0.25% of trypsin in a PBSsaline buffer free of calcium and of magnesium for 15 minutes at 37° C.

The cells are centrifuged at 200 g for 5 minutes in DMEM medium with 20%FCS for the trypsin inactivation. The cells are then filtered on nylonmembrane (pore size: 48 microns) and collected in a chemically definedmedium free of serum containing a 1/1 mixture of DMEM and Ham's F12medium supplemented with glutamine (4 mM), glucose (33 mM), penicillin G(50 U/ml), streptomycin sulfate (50 μg/ml) and an N2 supplement ofBottenstein and Sato (1979): putrescine (100 μM), sodium selenite (30nM), human transferrin (50 μg/ml), progesterone (20 nM), insulin (5μg/ml) and β-estradiol (1 pM). All the N2 supplements were bought fromSigma.

The cells are plated at a density of 7.5×10⁴ cells/cm² on 24-wellplastic plates. For some experiments, the plastic plates are coatedeither with fibronectin (24 μg/ml) or with thrombospondin (20 μg/ml).The cultures are incubated at 37° C. and under air containing 10% CO₂.The medium is not changed during the experiment. These cultures consistof nearly 95% of neurons.

Cell Cultures of Spinal Neurons

The spinal cords of 6-day old chicken embryos are dissected, freed oftheir meningeal membrane and cut into small pieces in a phosphate buffer(PBS) free of calcium and of magnesium. After incubation with 0.25%trypsin for 10 minutes at 37° C., the tissue is centrifuged at 200 g for4 minutes in a growth medium containing 20% fetal calf serum in order tostop the trypsinization. The cells are then dissociated by repeatedtrituration using a Pasteur pipette and resuspended in a chemicallydefined medium free of serum as above.

The cells are plated at a density of 7.5×10⁴ cells/cm² on 24-wellplastic culture plates. The cultures are incubated at 37° C. and underair containing 10% CO₂. The medium is not changed during the experimentsand it has already been shown that this type of cell populationcontained more than 93% of neurons.

The peptides tested are, in addition to the peptide according to thepresent invention mentioned above (peptide SEQ ID NO: 8), a secondpeptide according to the invention having the structure:W-G-P-C-S-V-S-C-G- (SEQ ID NO: 11)

-   -   then 3 peptides for comparison:        D-C-K-D-G-S-D-E- (SEQ ID NO: 12)        R-K-A-R- (SEQ ID NO: 13)    -   and a mixed sequence of the peptide SEQ ID NO: 8:        S-S-C-R-S-G-C-W-G-S-S-W- (SEQ ID NO: 14).

All these peptides were obtained by synthesis.

Results

In the presence of the peptide SEQ ID NO: 8, the neurons aggregate andare essentially connected by bundles of long and thick neurites after 5days of culture. Furthermore, these cells adhere well to the substratecoated with the peptide with no detachment of the aggregates. Bycontrast, the control cell cultures, in the absence of the peptide,rapidly detach from the plastic substrate at 5 days of culture. However,on plastic, only the cortical neurons form aggregates from which veryfew neurites can be observed, which indicates that the substrate isinsufficiently adhesive. The number of neuronal aggregates increases by9.3% between the control culture and the culture treated with thepeptide according to the invention.

Morphometric analysis reveals a significant increase both in the numberof neurites per aggregate and in the length of the neurites peraggregate. Moreover, wells of plastic coated with BSA are only veryslightly adhesive for the neuronal cells.

The tests carried out with other peptides in comparison with the peptideSEQ ID NO: 8 at random give no significant result.

The peptide SEQ ID NO: 11 gives lower but, nevertheless, significantresults.

Likewise, the tests carried out with the peptide SEQ ID NO: 13, which isa consensus sequence for attachment of glycosaminoglycans which ispresent in a large number of proteins which bind to heparin, as well asthe peptides corresponding to type A LDL receptors, gave norepresentative result.

Moreover, the effect of the peptides according to the present inventionSEQ ID NO: 8 and NO: 11 on cultures at low density was studied. Indeed,it has already been demonstrated that high aggregation could influenceneuritic growth in the same manner as the strength of adhesion of thecells to the substrate.

The tests carried out at low density showed that in the absence ofaggregation, the two peptides significantly increased the percentage ofneuronal cells carrying neurites. In the controls, only 24.4% of theadherent cells had neurites at 4 days of culture whereas 2 and 2.5 timesas many appeared in the presence of the peptides SEQ ID NO: 8 and NO:11, respectively.

The morphometric analyses revealed a significant increase in each ofthem both in the number of neurites per cell and the length of theneurites in the presence of the peptide SEQ ID NO: 8 and not the peptideSEQ ID NO: 11. Under these conditions, this demonstrates that, even inthe absence of neuronal. aggregation, the peptide SEQ ID NO: 8 and to alesser degree the peptide SEQ ID NO: 11 are capable of promoting theadhesion and the neuritic growth of the cortical neuronal cells.

The effect of the peptide SEQ ID NO: 8 of the invention was also studiedunder various experimental conditions:

In the presence of various substrates, it was possible to demonstrate,for example, that the peptide according to the invention significantlyincreased the number of neurites per aggregate in well-containing platescoated with thrombospondin and fibronectin, compared with the controls,as well as the length of the neurites per aggregate.

The activity of the peptide SEQ ID NO: 8 on the spinal cord cellcultures compared with controls shows that the neurons remaindistributed for at least one week in vitro. The neurons show prominentneuritic growths forming a network without fasciculation of theneurites. An increase in the number of synaptic contacts between theneurites is observed. By contrast, the neuronal cells of the controlsform, in general, small aggregates interconnected by long filaments. Theneurites growing from the aggregates form relatively rigid bundles alongwhich essentially simple, bi- or tripolar neurons can be seen.

The other peptides tested under the same conditions show no notabledifference compared with the controls.

EXAMPLE 2 Effect of the Peptide SEQ ID NO: 8 on the NeuroblastomaDerived from NIB104

Materials and Method

The cells derived from the NIB104 neuroblastoma were cultured in 24-wellplastic plates previously coated with a film of poly-L-lysine, underconditions similar to those for the primary cultures.

Results

In the presence of the peptide SEQ ID NO: 8 according to the presentinvention, the NIB104 neuroblastoma cells are considerably less numerousthan in the control cultures. The appearance of the cells isconsiderably modified because they acquire a characteristic neuronalphenotype. Morphometric analysis reveals that in the presence ofincreasing concentrations of peptide in the culture medium, the neuriticgrowth gradually increases. This response is therefore dose-dependantand indicative of a specific physiological effect.

1. A composition comprising the peptide: Y-W-S-A₁-C-S-A₂-C-G-Z(SEQ ID NO: 9) wherein Y and Z consist of: (a) amino acid chains consisting of 0–6 amino acids; or (b) chains of compounds which are not amino acids, wherein A₁ and A₂ are amino acid sequences consisting of 1 to 5 amino acids and further wherein the peptide is not selected from the group consisting of one of the following sequences: -W-S-P-C-S-V-T-C-G- (SEQ ID NO:2), -W-S-S-C-S-V-T-C-G- (SEQ ID NO:3), and -W-S-Q-C-S-V-T-C-G- (SEQ ID NO:4).
 2. The composition according to claim 1, wherein said A₁ is Pro or -X₁-W-X₂-X₃-(SEQ ID NO:5).
 3. The composition according to claim 2, wherein said A₁ is -X₁-W-S-X₃-(SEQ ID NO:6).
 4. The composition according to claim 1, wherein said A₂ is selected from the group consisting of -R-S-, -V-S-, and -V-T-.
 5. The composition according to claim 3, wherein said peptide is -W-S-X₁-W-S-X₃-C-S- A₂-C-G- (SEQ ID NO:7).
 6. The composition according to claim 4, wherein said peptide is: -W-S-G-W-S-S-C-S-R-S-C-G- (SEQ ID NO:8).
 7. A composition comprising a peptide according to claim 1 and a pharmaceutically acceptable vehicle.
 8. An additive for culturing nerve cells, comprising a peptide according to claim
 1. 9. A composition according to claim 1, wherein the peptide is selected from the group consisting of SEQ ID NOS: 97–168.
 10. A composition according to claim 2, wherein the peptide is selected from the group consisting of SEQ ID NOS: 50–59.
 11. A composition according to claim 3, wherein the peptide is selected from the group consisting of SEQ ID NOS: 60–64.
 12. A composition according to claim 4, wherein the peptide is selected from the group consisting of SEQ ID NOS: 65–88.
 13. A composition according to claim 5, wherein the peptide is selected from the group consisting of SEQ ID NOS: 89–96.
 14. A peptide consisting of the following amino acid sequence: -W-S-G-W-S-S-C-S-R-S-C-G- (SEQ ID NO:8).
 15. A composition comprising a peptide according to claim 14 and a pharmaceutically acceptable vehicle.
 16. An additive for culturing nerve cells, comprising a peptide according to claim
 14. 